EFFECT OF THERMAL PROCESSING ON COLIFORMS IN MINCED BEEF AND THEIR PUBLIC HEALTH SIGNIFICANCE IN PORT-SAID CITY

Dept. of Food Hygiene, Port-Said Lab.,

Animal Health Research Institute, Dokki, Giza, Egypt.

Effect of Thermal Processing

on Coliforms in Minced Beef and Their public Health Significance

in Port-Said City

 (With 4 Tables and One Figure)

Hassan El-S.M. Farag  andNahla  T. Korashy*

* Dept. of Microbiology, Port-Said Lab., Animal Health Research Institute,

 Dokki, Giza, Egypt.

(Received at 19/12/2006)

تأثير المعاملات الحرارية علي ميکروبات الکولي فورم المتواجدة في اللحم البقري المفروم وعلاقتها بالصحة العامة في مدينة بورسعيد

حسن السيد محمد فرج ، نهلة طه عبد الجواد قرشي

في دراسة لتحديد مدي تأثير المعاملات الحرارية علي ميکروبات الکولي فورم المتواجدة في اللحم البقري المفروم تم فحص خمسون عينة صالحة ظاهريا والتي تم جمعها عشوائيا من محلات الجزارة بمدينة بورسعيد بهدف عد وعزل وتصنيف ميکروبات الکولي فورم المتواجدة في هذه العينات قبل و بعد المعاملات الحرارية بالاضافة لمناقشة اثرهذه الميکروبات علي الصحة العامة. وأظهرت النتائج ان نسبة العينات الايجابية لميکروبات الکولي فورم الکلية ، الکولي فورم البرازية و ميکروب الاشريشيا کولاي کانت       100% (50)، 100 % (50) و 66 % (33) في اللحم البقري المفروم الطازج علي التوالي بينما کانت 82% (41)، 74 % (37) و50 % (25) في العينات المشوية      و64 % (32)، 60 % (30) و 34 % (17) في العينات المقلية علي التوالي. وکان متوسط العدد الکلي لميکروبات الکولي فورم الکلية 4.6X ³10 , 1.5X ³10              و 5.8X ²10 خلية/ جرام ومتوسط العدد الکلي لميکروبات الکولي فورم البرازية کان 1.1X ³10 , 5.1X ²10 و 2.6X ²10 خلية/ جرام بينما کان متوسط العدد الکلي لميکروب الاشريشيا کولاي 5.8X ²10 , 2.1X ²10 و 4.8X ¹10 خلية/ جرام  وذلک في عينات اللحم البقري المفروم الطازج و المشوي و المقلي على التوالي. وتم عد و تصنيف العترات المعزولة فوجد أن عدد عترات ميکروبات الکولي فورم الکلية 88، 54 و32 بينما کانت عترات ميکروبات الکولي فورم البرازية 63، 44 و 31 وکان عدد عترات ميکروب الاشريشيا کولاي 33، 25 و 17 وذلک في عينات اللحم البقري المفروم الطازج والمشوي والمقلي على التوالي. وبتصنيف الانواع المعزولة من العينات موضع الدراسة وجد ان العترات المعزولة کانت انواع من الاشريشيا کولاي ، انتيروبکتر کلواکا، انتيروبکتر ايروجينز، ستروباکتر فرونداي، ستروباکتر کوزيراي، سيريتيا مارسيسينز. وتم مناقشة تاثير المعاملات الحرارية علي ميکروبات الکولي فورم المتواجدة في عينات اللحم البقري المفروم موضع الدراسة.

SUMMARY

Fifty samples of apparently healthy minced beef were randomly purchased from butcher's shops of Port-Said city. The samples were examined for enumeration, isolation and identification of total coliforms, fecal coliforms and Escherichia coli before and after thermal treatment. The incidence of positive fresh minced beef samples for total coliform, fecal coliform and Escherichia coliwere 100% (50), 100% (50) and 66% (33), while that of roasting samples were  82% (41), 74% (37) and 50% (25) but the incidence of pan frying minced beef samples were  64% (32), 60% (30) and 34% (17) respectively. The mean values of the total coliform counts were 4.6 X 10³,1.5 X 10³ and 5.8 X 10² MPN/g and that of fecal coliform counts were 1.1 X 10³, 5.1 X 10² and             2.6 X 10² MPN/g in fresh, roasting and pan frying minced beef respectively. Meanwhile, the mean values of the Escherichia coli counts were 5.8 X 10², 2.1 X 10² and 4.8 X 10¹ MPN/g in fresh, roasting and pan frying minced beef respectively. The numbers of total coliform isolates were 88, 54 and 32 and that of fecal coliform were 63, 44 and 31 and the number of Escherichia coli isolates were 33, 25 and 17 for fresh, roasting and pan frying minced beef samples respectively. The bacterial isolates in the examined samples were identified as Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Citrobacter koseri and Serratia marcescens. The effect of thermal processing on total and fecal coliforms and Escherichia coli in the examined samples were discussed.

Key words: Meat products, minced beef, coliforms, thermal processing

Introduction

Minced beef is the largest selling and popular beef items in most markets. Besides to their palatability it has very high value protein and valuable quantities of B vitamins (Kalalou et al., 2004).

Minced meat provides a favorable environment for the growth of bacteria which normally found on the meat surface. These bacteria were distributed throughout the entire product during grinding and mixing processes used in fabrication of minced meat. Also minced meat may be subjected to the contamination directly by coliforms group from the faecal contamination which may be occur during slaughtering, evisceration and transportation (Emswiler et al., 1976; Hamdy et al., 1989; Rice et al., 1997).

Coliform is a group of bacteria inhabit the intestinal tract of worm blooded animals including man. Some species of them are opportunistic pathogens responsible for a wide range of infections while many other species of them are normally free living saprophytic. These bacteria are Gram-negative, aerobic and facultatively anaerobic bacteria, non spore forming and rod shaped, motile by flagella except Klebsiella (non motile), capable of fermenting lactose with the production of acid and gas includes Escherichia coli and species from the genera Escherichia, Klebsiella, Enterobacter, Serratia and Citrobacter (Greenberg and Hunt, 1985; FAO, 1992; Hitchins et al., 1998; FDA 2002).

Due to the heating process was not enough to ensure the safe eating of minced meat (Bayhan et al., 1990), so a lack of good sanitary practices in the production of minced meat may results in a meat of less keeping quality, increase the possibility of spoilage and causing a major health problem (Emswiler et al., 1976; Lochowich, 1978).

The health problem of coliform group depends upon the production of different toxin and colonization factors. The enterotoxigenic strains of Klebsiella, Escherichia, Enterobacter, Serratia and Citrobacter have been isolated from infants and children with acute gastroenteritis. These toxins are heat labile and heat stable types and have other properties in common with Escherichia coli toxins. The endotoxins of most coliforms show a bacteraemia in human characterized by fever, vital organs hypoperfusion, depletion of complement, hypotension, irreversible shock and death. Coliforms rarely cause extra-intestinal diseases as bacteraemia and meningitis unless host defense are compromised (Guentzel, 1982; APHA, 1984).

 The most virulent and major enteric pathogenic group particularly in developing countries was Enterovirulent Escherichia coli group (EEC) which implicated in food born illness and includes enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), enteroinvasive Escherichia coli (EIEC) (Hitchins et al., 1998; FDA, 2002). These groups have been incriminated in many cases of food borne disease outbreaks, travelers' diarrhea, infantile diarrhea and colibacillosis in adults. There are two forms of the disease which differ clinically from each other to some degree. The first form caused by enteropathogenic strain, is characterized by excessive loss of fluid from profuse diarrhea (Cholera like syndrome) (Pickering, 1986; Nataro and Kaper, 1998). The second form tend to reflect toxins, heat stable toxin (ST) or heat labile toxin (LT) leading to food poisoning, possible accompanied by nausea, fever, abdominal cramps, vomiting, watery or bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombocytopenic purpura (Karmali, 1989; Reed, 1994; Acheson et al., 1996; Ward et al., 1997).

Thus the main purpose of this work was undertaken to evaluate the effect of thermal processing on coliforms in minced beef besides the study of their public health significance in Port-Said city.

Materials and methods

1: Samples collection:

A total of 50 apparently healthy samples of minced beefwere randomly collected from butcher's shops from Port-Said city. Each individual sample (250 g) was placed separately into sealed sterile plastic bag, thoroughly identified and delivered to the laboratory in a refrigerated container at 4^°C. All specimens were prepared, treated and examined within 4 hours of collection. The samples were divided into 2 parts; the 1^st part weight 50 g directed to counting, isolation and identification of coliforms and Escherichia coli. The 2^nd part (200 g) was thermally treated according to the approved processing technique before the bacteriological examination.  

2: Bacteriological examination:

2-1: Preparation of the samples:

A representative 50 g sample of minced beef(fresh or thermally treated) were taken aseptically and homogenized in 450 ml bufferfield's phosphate buffer and blend for 2 min. at 10000-12000 rpm. Then tenfold serial dilution was prepared using bufferfield's phosphate buffer till dilution 10⁵ according to FAO (1992).

2-2: Presumptive test for coliforms: 

Three tubes each containing 10 ml of sterile lauryl tryptose broth (LT) with Durham's tubes, were inoculated with 1 ml from each of the original dilution. Inoculated tubes were thoroughly mixed before being incubated in a thermostatically controlled incubator at 35^°C for 48 hours. The inspection was done after 24 and 48 hours incubation for positive gas production (FAO, 1992).

2-3: Probable number of total coliforms:

Gently agitate each positive LT tube and transfer loopful of suspension to tube of brilliant green bile broth 2% (BGB) containing inverted Durham's tube. All broth tubes were incubated at 35 ^°C for         48 ± 2 hr and examined for gas production. Enumeration of total coliforms was applied by using table of most probable number (MPN) based on combination of confirmed gassing LT tubes for 3 consecutive dilutions.

2-4: Isolation of total coliforms:

            Streak loopful of suspension from gassing LT tube to xylose lysine desoxycholate agar (XLD) and incubate at 35^°C for 18-24 hr according to FAO (1992).

2-5: Probable number of fecal coliforms:

Gently agitate each gassing LT tube and transfer loopful of suspension to tube of EC medium containing inverted Durham's tube. All broth tubes were incubated at 45.5 ± 0.2^°C for 48 ± 2 hr. The tubes were examined at 24 hr for positive gas production and re-incubate the negative tube for additional 24 hr and examined 2^nd time for gas production. Enumeration of fecal coliforms was applied by using table of most probable number (MPN) based on proportion of confirmed gassing EC medium tubes for 3 consecutive dilutions.

2-6: Isolation of fecal coliforms:

            Streak loopful of suspension from gassing EC medium tubes to xylose lysine desoxycholate agar (XLD) and incubate at 35^°C for 18-24 hr according to FAO (1992).

2-7: Enumeration of Escherichia coli:

Streak loopful of suspension from gassing EC medium tubes to    L-EMB (Levine's Eosin-Methylene Blue) agar plate and incubate at 35^°C for 18-24 hr. Then examined the plate for the typical Escherichia coli (dark center with or without metallic sheen). Enumeration of Escherichia coli were applied by using table of MPN based on proportion of EC medium tubes in 3 consecutive dilutions which shown to contain Escherichia coli according to FAO (1992).

2-8: Biochemical identification of the isolates:

Pick three typical colonies from each agar plate and transfer to plate count agar (PCA) slants tube and incubate at 35^°C for 18-24 hr according to FAO (1992). All PCA slants were directed to morphological and biochemical identification according to Brenner (1984); Holt et al. (1994) and Farmer (1995).

3: Experimental thermal processing:

The 2^nd part (200 g) was divided into 2 parts (100 g for each part), one part for the conventional roasting technique and the other for pan frying technique.

4- Statistical methods

Minimum, maximum, mean, standard deviation and standard error of mean and frequency distribution were used to describe data. T-test was used to evaluate the significant effect of the different methods of thermal processing on the total and fecal coliforms and Escherichia coli. P value was considered significant if less than 0.05 and 0.01 at 95% and 99% respectively. These tests were analyzed using the Statistical Package for Social Scientists (SPSS) for windows 12.0 (SPSS Inc., Chicago, IL, and USA).

Results

Table 1: Statistical analytical results of the total coliform, fecal coliform and Escherichia coli counts (MPN/g) recovered from fresh and thermal processed minced beef.

ND= Non-Detectable (<30).   D= Detectable (>30).  MPN = Most Probable Number

Min. = Minimum.  Max. = Maximum.  SE = Standard Error    SD = Standard Deviation.

Table 2: Frequency distribution of the examined fresh and thermal processed minced beef based on their total coliform, fecal coliform and Escherichia coli counts (n=50 of each).

< 30 = Non detectable level.

Table 3: Correlation between total coliform, fecal coliform and Escherichia coli counts based on the examined fresh and thermal processed minced beef (n=50 of each).

(**) = Highly significant correlation at <0.01 (99% confidence) by using t-test (Paired system test).

(*) = Significant correlation at <0.05 (95% confidence) by using t-test (Paired system test).

E. coli = Escherichia coli

Table 4: Incidence of coliforms isolates recovered from fresh and thermal processed minced beef.

Discussion

Members of the coliform groups are referred as general indicator microorganisms to measure the potential presence of enteric pathogens (for example Escherichia coli) in foods, besides the measuring of fecal contamination of the food products and the sanitary condition in the food-processing environment (Greenberg and Hunt, 1985; APHA, 1992; FAO, 1992). The possibility of food health problem by coliforms depending upon the number, types and the virulent of these pathogens whereas, the higher the number, the greater the possibility of pathogenic organisms being present (FAO, 1992).

The given results in Table (1) revealed that the incidences of positive fresh minced beef samples for total and fecal coliforms and Escherichia coli were 100% (50), 100% (50) and 66% (33) respectively. The incidence of positive samples for coliforms was higher than the results recorded by (Kaloianov et al., 1987; Willshaw et al., 1993; Heredia et al., 2001; Tanios et al., 2002; Cohen et al., 2006) but that of Escherichia coli was lower than those recorded by (Heredia et al., 2001). The high incidence of positive samples may be attributed to the unsanitary conditions during the production and manufactured of minced beef (Wilson, 2001). The mean values of the total and fecal coliforms and Escherichia coli counts in fresh minced meat were 4.6 X 10³, 1.1 X 10³ and 5.8 X 10² (MPN/g) in the examined fresh minced beef respectively (Table 1). These results agreed with the results recorded by (Rogers and McCleskey, 1957; Küpeli, 1996) but lower than the coliforms results recorded by (Yilmaz et al., 2002a). On the other hand our results were higher than the results recorded by (Kaloianov et al., 1987; Yilmaz et al., 2002; Firstenberg et al., 2004). The high figures of our results may be attributed to contamination of meat during slaughtering, evisceration and transportation (Hamdy et al., 1989), bad microbiological quality and heavy load coliform of trimming and cuts of beef (Emswiler et al., 1976; Bayhan et al., 1990), unsanitary conditions during the manufacture (APHA 1992; Wilson, 2001) and after manufacture as bad handling and packaging of minced beef (Yilmaz et al., 2005; Cohen et al., 2006), besides long chilling storage or holding of the minced beef at an inappropriate temperature (Kandeepan and Biswas, 2005; Yilmaz et al., 2005). Meanwhile the higher counts of fecal coliform and Escherichia coli may be attributed to recent fecal contamination (Caplenas and Kanarek, 1984; Greenberg and Hunt, 1985; APHA, 1992; FDA, 2002) from contaminated or infected cattle (Rice et al., 1997) with the upper mention causes.

The presented results in Table (2) showed that the frequency distribution of the total and fecal coliforms and Escherichia coli within the range 30 - <10⁴ were 74% (37), 100% (50) and 66% (33) respectively but 26% (13) of the fresh minced beef showed the total coliforms with the range ≥10⁴ MPN/g and 34% (17) of the examined fresh minced beef had non detectable levels (< 30 MPN/g) for Escherichia coli. These results were higher than the results recoded by (Surkiewicz et al., 1975; Firstenberg et al., 2004). Higher results may be attributed to heavy load and high initial counts of coliforms and Escherichia coli (Bayhan et al., 1990) and unsanitary conditions during the manufacture of minced beef with long chilling storage (Wilson, 2001).

            Regarding to the effect of thermal processing on total and fecal coliforms and Escherichia coli presented in Table (1) It is evident that the incidences of positive roasting minced beef samples for total and fecal coliforms and Escherichia coli were 82% (41), 74% (37) and 50% (25) respectively while in case of the examined pan frying minced beef the incidence were decreased to 64% (32), 30% (30) and 34% (17) respectively. On the other hand the mean values of the total and fecal coliforms and Escherichia coli counts were 1.5 X 10³, 5.1 X 10² and 2.1 X 10² MPN/g for roasting minced beef respectively while that of pan frying minced beef were reduced to 5.8 X 10², 2.6 X 10² and 4.8 X 10¹ MPN/g under effect of roasting technique respectively.

            Also the thermal processing effect presented in Table (2) revealed that the percentage of the examined samples showed non detectable levels (< 30 MPN/g) for total coliform, fecal coliform and Escherichia coli under effect of roasting technique were 18% (9), 26% (13) and 50% (25) respectively, while under the effect of pan frying technique the percentage of the examined samples showed non detectable levels (< 30 MPN/g) for total coliform, fecal coliform and Escherichia coli were increased to 36% (18), 40% (20) and 66% (33) respectively. On the other hand most of the examined roasting minced beef samples [82% (41), 74% (37) and 50% (25)] had a rang of 30 - <10⁴ MPN/g for total and fecal coliforms and Escherichia coli while that of pan frying minced beef samples [50% (25), 60% (30) and 34% (17)] were reduced to 30 - <10³ MPN/g respectively. These results with Fig (1) showed that the heat processing either roasting or pan frying technique reduce the total and fecal coliforms and Escherichia coli counts to a different levels. This reduction effect agrees with the result recorded by (Delchev et al., 1978; Jay, 1978; Zahra et al., 1985; Desmarchelier and Grau, 1997; Yilmaz et al., 2005) but this reduction was impossible to eliminate all forms of coliforms (Jay, 1978).

Statistically by using t-test, showed a highly significant relationship between the counts of each of total and fecal coliforms and Escherichia coli recovered from fresh minced beef samples with that of the pan frying samples but roasting technique showed a significant relationship between them (Table 3). On the other hand the relationship between pan frying and roasting technique showed a significant relationship. This mean pan frying technique was more effective and more efficient than roasting technique in reduction effect (Yilmaz et al., 2002).

The obtained results in Table (4) showed that the number of total and fecal coliforms and Escherichia coli isolates of fresh minced beef were 88, 63 and 33 isolates respectively. In case of roasting technique, these isolates were reduced to 54, 44 and 25 for total and fecal coliforms and Escherichia coli respectively while in case of pan frying minced beef samples the isolates were more reduced to 32, 31 and 17 isolates respectively. The coliforms organisms recovered from the examined samples were identified as Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Citrobacter koseri and Serratia marcescens. The main and important isolate found in fresh and treated samples was Escherichia coli. On the other hand Citrobacter freundii, Citrobacter koseri and Serratia marcescens found only in total coliform and this may be attributed to that these group of microorganisms can not tolerate high temperature but fecal coliform and Escherichia coli can tolerate and considered thermotolerant organisms (Caplenas and Kanarek, 1984). The reduction in the number of isolates was more in pan frying than in roasting technique and this agrees with the result recorded by (Zahra et al., 1985) who showed that coliforms were easily killed (Jay, 1978) and decreased markedly after heat treatment but heating process was not enough to ensure the safe eating quality (Bayhan et al., 1990) and impossible to eliminates all forms of coliforms (Jay, 1978). The variation between our result and the other results recorded by (Yilmaz et al., 2005) may attributed to the variation in season and locality (Cohen et al., 2006) and the high initial counts of coliforms found in minced beef (Zahra et al., 1985).        

The safety of minced meat associated with the keeping quality of meat cuts, sanitary condition during handling, fabrication, packaging, equipment and utensils used and time/temperature of storage (Emswiler et al., 1976; APHA, 1992). The endotoxins of coliform groups, heat labile and heat stable types specially that of Escherichia coli toxins (Pickering, 1986; Hitchins et al., 1998) with the undercooked or improperly cooked minced beef and cross and post cooking contamination by these microorganisms (Guentzel, 1982; Ward et al., 1997; Cohen et al., 2006) results in a major health problem to the consumers. Consequently the presence of large number of coliforms in foods is highly undesirable but it would be almost impossible to eliminate all forms of coliform (Jay, 1978). In conclusion, to increase the shelf life and the safety of minced meat and decrease their coliforms loads and prevention the infections by these microorganisms to the consumers, a groups of measurement should be attempt such as good and strictly hygienic measurement pre and during slaughtering, evisceration and transportation with the avoidances of the contamination of meat from other infected or contaminated animals. Also personal hygiene and removal the source of pollution as fecal contamination and sewage water with good sanitary condition during handling, grinding and mixing processes besides the equipment and utensils used in fabrication produce a minced beef of good keeping quality. The refrigeration (below 4°C) and good time/temperature storage with sufficiently cooking of minced meat and prevent the post cooking cross contamination and prohibiting people who are ill from working in food operations and strictly prevention the consumption of insufficient cooked minced meat specially in compromised hosts reduce the consumers health problem.

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